2.9. Immunofluorescence Staining and BrdU Staining

For immunofluorescence analysis of the cultured neurosphere or cells, cells were fixed in ice-cold 4% paraformaldehyde for 15 minutes and then washed three times in 0.01 M phosphate-buffered saline (PBS) (pH 7.4). For immunofluorescence analysis of the brain, brain slices were obtained following the procedures in the previous article [22]. After blocking using serum, brain slices or cultured cells were incubated with the primary antibodies at 4°C overnight. The antibodies used in this study are listed as follows: anti-Nestin (1 : 200; SAB4200347, Sigma-Aldrich, USA), anti-β-III-tubulin (Tuj1; 1 : 500; MAB1637, Sigma-Aldrich, USA), anti-NF-200 (1 : 200; N0142, Sigma-Aldrich, USA), anti-ChAT (1 : 200; A01192-4, Boster, Wuhan, China), and anti-GFAP (1 : 200; sc-33673, Santa Cruz, USA).The sections were then incubated with Alexa Fluor second antibody (1 : 200; Thermo Fisher Scientific, USA) for 2 h and counterstained by Hoechst 33342 (C1022, Beyotime, Shanghai, China) for 20 mins. The sections were then cover slipped with mounting medium (ZSGB-Bio, Beijing, China).

The proliferative activity of NSCs and verification of NSCs after transplantation were investigated by BrdU labeling and staining. Cultured cells or brain sections were treated with 2 M HCl, at 37°C for 30 mins to denature DNA, and added in 0.1 M borate buffer (pH 8.5) for 10 mins. Sections were then incubated with anti-BrdU antibody (1 : 200; B2531, Sigma-Aldrich, USA) and Alexa Fluor second antibody. Fluorescent signals were visualized by the confocal laser microscope system (TCS SP5, Leica, Germany).